Recent observations by the grant applicant and others indicate that host-derived fibroblasts actively participate in chronic rejection (CR) of allografts. The new finding could impact human health care in a positive way: if the fibroblast progenitors (FP) are defined, new strategy aiming at manipulating their migration and proliferation will become possible. In order to appreciate this potential, we must first identify and characterize the mesenchymal progenitors that give rise to intragrafl fibroblast (IF), and must determine the mechanisms by which FP migrate into allografts. The central hypothesis is that proliferating IF are derived from FP of the allograft recipient. The FP are committed to fibroblastic lineage in the bone marrow (BM), released into the blood circulation and attracted by local factors to migrate into the aliograft via interaction of cell surface CD44 to hyaluronan of the extracellular matrices (EMC). Two Specific Aims will be pursued: (1) Define the progenitor cells of intragraft fibroblasts. Clonal analysis, differentiation cultures and FACS analysis of cell surface antigens will be conducted on MSC populations derived from rat BM and fibroblasts of cardiac allografls to isolate and define the FP. Defined clonal FP will be vector-marked and engrafted into rat recipients of allograft for assessing their ability to migrate into the cardiac allografts and become IF. (2) Determine the migratory mechanisms that are responsible for the recruitment of fibroblast progenitors in allografts. A model of MSC migration to allograft that is regulated by CD44/HA will be tested. Pro-inflammatory factors will be determined for their ability to up-regulate CD44 expression by MSC. The CD44v isoforms used by migrating FP will be identified using real time RTPCR and nucleotide sequencing. CD44 null MSC will be retrovirally transduced to express selected CD44v isoform(s) and the transduced MSC will be tested for HA binding and migratory ability in experimental animal model. Completion of this research project will have defined the FP of IF and determined the migratory mechanisms by which FP home to allografts during chronic rejection.